Journal
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Volume 436, Issue 3, Pages 430-435Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2013.05.121
Keywords
NFkB; p50/p65; Dissociation constant; Heterodimer; Fluorescence measurement; FCS/FCCS; Live cell imaging
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Funding
- JSPS [21221006]
- MEXT [19058001]
- Grants-in-Aid for Scientific Research [19058001, 21221006] Funding Source: KAKEN
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Two-laser-beam fluorescence cross-correlation spectroscopy (FCCS) is promising technique that provides quantitative information about the interactions of biomolecules. The p50/p65 heterodimer is the most abundant and well understood of the NF kappa B dimers in most cells. However, the quantitative value of affinity, namely the K-d, for the heterodimer in living cells is not known yet. To quantify the heterodimerization of the IPT domain of p50/p65 in the living cell, we used two-laser-beam FCCS. The K-d values of mCherry(2)- and EGFP-fused p50 and p65 were determined to be 0.46 mu M in the cytoplasm and 1.06 mu M in the nucleus of the living cell. These results suggest the different binding affinities of the p50/p65 heterodimer in the cytoplasm and nucleus of the living cell and different complex formation in each region. (C) 2013 Elsevier Inc. All rights reserved.
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